This is a renewal of a highly synergistic PPG exploring tolerance induction for xenotransplantation from pigs. Themes include: 1) Tolerance in the adaptive immune system; 2) Overcoming innate immune barriers; and 3) Using CRISPR/Cas9 to improve genetically modified (GM) MGH inbred miniature swine. In Project 1, Achieving Xenograft Tolerance through Thymic Programming in Primates, we will understand and avoid proteinuria, which limits the survival of baboons receiving GalT KO pig kidney transplants. We will optimize tolerance and protective immunity by transplanting a hybrid vascularized thymus (VT) containing pig and baboon TECs and combine this with mixed xenogeneic chimerism with intra-bone injection of pig bone marrow to simultaneously tolerize adaptive and innate immunity. Project 2, Achieving Xenograft Tolerance through Mixed Chimerism, also uses the pig-to-baboon model. We have achieved markedly prolonged mixed chimerism in baboon recipients of human CD47 (hCD47) Tg pig hematopoietic cells (HCs), resulting in remarkably prolonged pig skin graft survival. We will combine this approach with intrabone injection of hCD47 Tg/hCD55 Tg/GalT KO pig hematopoietic cells and use ex-vivo expanded recipient Tregs to enhance engraftment, aiming to achieve durable chimerism and, with it, tolerance of B, NK and T cells. We will test HCs from new GM swine produced in Project 4 and will carry out high throughput discovery of genes to enhance pig HC survival in collaboration with Project 4. Project 3, Tolerance of Adaptive and Innate Human Anti-Pig Immune Responses in Humanized Mice, combines mixed xenogeneic chimerism and porcine thymic transplantation in humanized mice. We will combine engineered pig/human hybrid thymi with mixed xenogeneic chimerism to achieve global immune tolerance along with protective immunity for both human and porcine tissues. We will determine the impact of the hCD47 Tg on induction of porcine mixed chimerism, identify additional potential human innate immune barriers to porcine chimerism and assess the effect of new GMs on porcine chimerism in mice with human immune systems. In Project 4, Improving Xenogeneic Chimerism and Tolerance through Genome Engineering Technology, CRISPR/Cas9 will be used to generate optimal GM pigs for tolerance induction and organ transplantation. We will also generate libraries of constructs that will be used to transduce pig LCLs for high-throughput screening in vivo to identify GMs that enhance survival in baboons. This novel approach will lead to future porcine GMs that would not have been considered using a more biased approach. Core A will provide administrative support for the PPG, facilitating interactions between the primary and subcontract sites. Core B will support all large animal needs, including non-human primates and GM MGH inbred miniature swine; provide support for infectious diseases, coagulation and clotting issues of swine and baboons; and provide mAbs and antisera. These studies, cores and interactions will synergize to provide major leaps forward toward clinical xenotransplantation.